I. Field of the Invention
This invention relates to glycolipids useful for serodiagnosis of tuberculosis and leprosy and to serodiagnosis techniques using such glycolipids. More particularly, the invention relates to synthetic pseudo cord factor-like glycolipids useful for these purposes.
II. Description of the Prior Art
Enzyme-linked immunosorbant assays (usually referred to as ELISA) and similar techniques (e.g. so-called "spot tests" which are a simplified form of ELISA test) for diagnosing diseases in human and animal patients have become very useful and popular in recent years because of their simplicity and their acceptable sensitivity and specificity. These techniques are based on the binding effects of antibodies and antigens. In one form of the ELISA assay, for example, an antigen produced by a specific organism is used to test for the presence of antibodies for the antigen in the sera of patients, thus providing an indication that the patients have been exposed to these organisms. The antigen is immobilized on a solid support and incubated with the serum to be tested. If a target antibody is present in the serum, indicating exposure of the patient to the disease-causing organism, it binds to the layer of antigen. The number of antigen/antibody bound molecular pairs produced in this way depends on the concentration of the antibody in the serum until saturation of the antigens in the layer takes place. After washing the layer attached to the support, a solution of an enzyme-linked antibody (e.g. goat-antihuman IgG) for the bound protein is contacted with the supported layer. After a second washing step, the layer is contacted with a solution of a substrate for the enzyme and the bound enzyme, if present, converts the substrate to a detectable product.
In the so-called "spot tests", the microtiter plate usually used as a solid support for the antigen in the ELISA test is replaced by a strip of paper (cellulose nitrate, etc.). The strip is spotted with the antigen and for instance Protein A is used instead of the conjugate and a colloidal gold solution in place of the substrate.
ELISA and spot tests of this kind have been developed for detecting a number of disease-producing organisms. However, a satisfactory test has not yet been developed for tuberculosis produced by the bacterium Mycobacterium tuberculosis and leprosy produced by the bacterium Mycobacterium leprae, the two most widespread mycobacterial diseases affecting mankind. The difficulty in developing suitable tests has resulted from the fact that M. tuberculosis and M. leprae produce large numbers of immune response-producing proteins, some of which appear to be common to other microorganisms that may or may not be pathogenic. Hence, positive test results produced by known antigens are generally unreliable (false positives) and other tests have to be carried out to confirm the presence of the tuberculosis or leprosy infections.
Hopes of developing a reliable ELISA test for tuberculosis were heightened recently by the discovery of an M. tuberculosis species-specific trehalose-glycolipid provisionally designated as "SL-IV" (F. Papa, et. al., "Serological Specificity of M. tuberculosis Glycolipids" (1989), Acta Leprologica 7 (Suppl. 1):98-101). An ELISA serological procedure using this protein has been shown to have good potential for the diagnosis of tuberculosis and leprosy. However, SL-IV is extracted from cultures of strains of M. tuberculosis and this extraction and the following purification procedures are difficult, time-consuming and expensive. The available quantities of this antigen are therefore quite limited and, moreover, the resulting antigen is a complex mixture of 2,3-trehalose esters. This makes SL-IV of rather limited use for widespread application in ELISA testing.
Trehalose-based glycolipids are found in a variety of structural forms in the lipids of mycobacteria and related bacteria. Serologically active glycolipids extracted from M. bovis BCG have been described (Reggiardo et. al., "Serologically Active Glycolipid Families From Mycobacterium bovis BCG", Am. J. Epidemiol. 10 (1975); 477-486). Three families of glycolipids called A, B and C reacted with sera from patients with tuberculosis and leprosy. Among the antigens studied, one designated Al gave the lowest incidence of false negative serological reactions and was later shown to be 6-0-mycoloyltrehalose (TMM). On the other hand, so-called "cord factor" (6,6'-di-O-mycoloyltrehalose) (TDM), does not seem to be useful as a coating antigen (Goren, M. B., "Mycobacterial Fatty Acid Esters of Sugars & Sulfosugars", Handbook of Lipid Research (Ed. D. J. Hanahan), Vol. 6 (Ed. M. Kates), Glycolipids, Phospholipids and Sulfoglycolipids, pp. 363-396, 1990). Again, however, even the potentially useful antigens have to be extracted from bacterial cultures with the attendant disadvantages.
Accordingly, there is still a need for improved antigens for use in enzyme-linked immunosorbant assays and similar tests for detecting tuberculosis and leprosy.